Ultrastructural evidence for the existence of actinorhizal symbioses in the late Pleistocene

Macrofossil actinorhizal root nodules discovered in sediments about 11 500 radiocarbon years old in northern Vermont, U.S.A., were studied by scanning electron microscopy. Evidence of an endophytic microsymbiont was observed within the cortex of the fossil nodules. A comparative study as undertaken using root nodules from species of Alnus, Dryas, Elaeagnus, Myrica, Shepherdia, and Vicia native to eastern North America in an effort to determine the identity of the fossils. From qualitative and quantitative differences observed among the root nodule endophytes, it was concluded that the fossil nodules contained actinomycetes morphologically similar to those of extant Elaeagnaceae. The fossils are probably from plants of Elaeagnus commutata and (or) Shepherdia canadensis based on independent evidence of the representation of these species in the plant macrofossil assemblage.

Effective and ineffective root nodules in Myrica faya

In cross-inoculation trials, inocula containing the nodule endophytes of Myrica gale, M. cerifera, M. cordifolia and M. pilulifera respectively were applied to the roots of young plants of M. faya Ait. growing in nitrogen-free culture solution. All four inocula induced nodule formation, and except where the M. gale inoculum had been used the nodules were of effective type and enabled the plants bearing them to grow nearly as well as other M. faya plants associated with the normal endophyte. The nodules induced by the M. gale endophyte were very numerous, but remained small and fixed no significant amount of nitrogen, and were thus ineffective. Light and electron microscopy showed that in the effective nodules induced by the normal endophyte or by that of M. cordifolia , the endophyte was confined to a layer 1-2 cells deep near the middle of the nodule cortex, and that in respect of the width of the hyphae and their production of club-shaped internally subdivided vesicles, the endophytes resembled closely those in the nodules of the few other species of Myrica that have been studied by modern methods of microscopy. In ineffective nodules the disposition of the infected cells was unchanged, but within the cells only a sparse development of the endophyte was observed, and no vesicles were found. The finding that nodules lacking vesicles showed little or no fixation is consistent with other evidence that the vesicles normally produced by non-legume nodule endophytes are the main site of nitrogen fixation.

Ultrastructure of the Alnus crispa var. mollis Fern. root nodule endophyte

Nitrogen-fixing, field-obtained root nodules of the silky green alder were studied by transmission electron microscopy. The nodule endophyte exhibited a prokaryotic cytology and was present in two forms: the hypha (0.3–1.0 μm), which was branched and septate, and the vesicle (3–5 μm), which was also septate and developed at the parental hypha tip. Bacteria-like cells, previously observed in light microscopy studies, were not seen in the present work. The actinomycete-like endophyte penetrated through the host cell wall and became enveloped by a capsular material (0.1 μm), the whole being enclosed by host membranes. In some host cells, the endophyte appeared to lyse and become a mass of shrunken debris. The fine structure of the Alnus crispa var. mollis root nodule endophyte was found to be similar to that of other non-leguminous root nodule endophytes.